Dept of Environmental and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, 485 E, Gray Street, Louisville, KY 40202, USA.
Proteome Sci. 2011 Jul 7;9:38. doi: 10.1186/1477-5956-9-38.
Reversed phase high performance liquid chromatography (HPLC) interfaced to electrospray tandem mass spectrometry (MS/MS) is commonly used for the identification of peptides from proteolytically cleaved proteins embedded in a polyacrylamide gel matrix as well as for metabolomics screening. HPLC separations are time consuming (30-60 min average), costly (columns and mobile phase reagents), and carry the risk of column carry over between samples. The use of a chip-based nano-ESI platform (Advion NanoMate) based on replaceable nano-tips for sample introduction eliminates sample cross-contamination, provides unchanging sample matrix, and enhances spray stability with attendant increases in reproducibility. Recent papers have established direct infusion nano-ESI-MS/MS utilizing the NanoMate for protein identification of gel spots based on full range MS scans with data dependent MS/MS. In a full range scan, discontinuous ion suppression due to sample matrix can impair identification of putative mass features of interest in both the proteomic and metabolomic workflows. In the current study, an extension of an established direct inject nano-ESI-MS/MS method is described that utilizes the mass filtering capability of an ion-trap for ion packet separation into four narrow mass ranges (50 amu overlap) with segment specific dynamic data dependent peak inclusion for MS/MS fragmentation (total acquisition time of 3 minutes). Comparison of this method with a more traditional nanoLC-MS/MS based protocol utilizing solvent/sample stream splitting to achieve nanoflow demonstrated comparable results for protein identification from polyacrylamide gel matrices. The advantages of this method include full automation, lack of cross-contamination, low cost, and high throughput.
反相高效液相色谱（HPLC）与电喷雾串联质谱（MS/MS）联用通常用于鉴定从聚丙烯酰胺凝胶基质中酶切的蛋白质中嵌入的肽，以及代谢组学筛选。HPLC 分离耗时（平均 30-60 分钟）、昂贵（柱和流动相试剂），并且存在柱间样品交叉污染的风险。基于可更换纳米尖端的芯片基纳喷雾平台（Advion NanoMate）用于样品引入，消除了样品交叉污染，提供了不变的样品基质，并通过提高喷雾稳定性来提高重现性。最近的论文已经建立了直接进样纳喷雾 MS/MS 利用 NanoMate 进行凝胶斑点的蛋白质鉴定，基于全范围 MS 扫描和数据依赖的 MS/MS。在全范围扫描中，由于样品基质的不连续离子抑制会损害在蛋白质组学和代谢组学工作流程中感兴趣的潜在质量特征的鉴定。在本研究中，描述了一种扩展的直接注入纳喷雾 MS/MS 方法，该方法利用离子阱的质量过滤能力将离子包分离成四个窄质量范围（50 amu 重叠），并针对 MS/MS 碎片化进行分段特定动态数据相关的峰包含（总采集时间为 3 分钟）。与利用溶剂/样品流分束实现纳流的更传统的纳 LC-MS/MS 基于协议的比较，该方法用于从聚丙烯酰胺凝胶基质中鉴定蛋白质的结果相当。该方法的优点包括全自动、无交叉污染、低成本和高通量。
